Patulin Ameliorates Hypertrophied Lipid Accumulation and Lipopolysaccharide-Induced Inflammatory Response by Modulating Mitochondrial Respiration

Patulin (PAT) is a natural mycotoxin found in decaying pome fruits. Although some toxicological studies have been conducted on PAT, recent research has highlighted its anticancer and antifungal effects. However, studies have yet to examine the effects and molecular mechanisms of PAT in other metabolic diseases. Obesity is a chronic disease caused by excessive food intake and abnormal lifestyle, leading to low-grade inflammation. Therefore, this study aimed to elucidate the effect of PAT on obesity at the cellular level. PAT treatment reduced lipid accumulation, suppressed glucose and LDL uptake, inhibited lipid deposition and triglyceride synthesis, upregulated fatty acid oxidation-related genes (Pgc1α), and downregulated adipogenic/lipogenic genes (Pparγ and C/ebpα) in hypertrophied 3T3-L1 adipocytes. Additionally, PAT treatment enhanced mitochondrial respiration and mass in differentiated adipocytes and alleviated inflammatory response in activated RAW 264.7 macrophages. Moreover, PAT treatment downregulated pro-inflammatory genes (il-6, Tnf-α, Cox-2, and inos), suppressed lipopolysaccharide (LPS)-induced increase in inflammatory mediators (IL-6, TNF-α, and NO), and restored mitochondrial oxidative function in LPS-stimulated macrophages by improving oxygen consumption and mitochondrial integrity and suppressing ROS generation. Overall, these findings suggest a potential for PAT in the prevention of lipid accumulation and inflammation-related disorders.


Introduction
Obesity represents a significant health issue marked by excessive fat accumulation, arising from an imbalance between energy consumption, storage, and expenditure, and is intricately associated with the onset of several metabolic disorders, including insulin resistance, diabetes, and cardiovascular disease [1,2].Additionally, obesity is mainly associated with increased white adipose tissue mass due to the activation of adipogenesis and elevated cytoplasmic triglyceride (TG) deposition [3].Adipogenesis is a tightly regulated process, in which undifferentiated preadipocytes differentiate into mature adipocytes [4].Adipogenesis is mainly governed by a complex network of transcription factors, including CCAAT/enhancer-binding protein (C/EBP) and peroxisome proliferator-activated receptorγ (PPAR-γ), which stimulate fatty acid synthesis and transport to promote TG accumulation in mature adipocytes [5].The activation of these transcription factors can upregulate the expression of key genes involved in lipid metabolism and adipocyte function, such as fatty acid synthase (FASN), fatty acid-binding protein 4 (FABP4), and ATP-citrate lyase (ACLY) [6].Additionally, adipocytes secrete various adipokines, including adiponectin, which are involved in insulin signaling and glucose regulation [7].The upregulation of adipogenesis and accumulation of mature adipocytes contribute to the disruption of fatty acid oxidation and glucose metabolism, subsequently compromising glucose homeostasis and insulin sensitivity [8].Therefore, modulating processes that control adipocyte differentiation could be an effective strategy to prevent or treat metabolic diseases.
Obesity is considered a low-grade chronic inflammatory disease due to an imbalance between pro-inflammatory stimuli and anti-inflammatory immune response [9].The release of pro-inflammatory molecules from adipose tissue is a significant mechanism that underlies the inflammatory nature of obesity [9].Specifically, the infiltration of macrophages into adipose tissue is responsible for the secretion of several substances known as adipokines, including pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) [10].Additionally, infiltrating immune cells in adipose tissue, particularly macrophages, interact with adipocytes and further contribute to the development of chronic low-grade inflammation [11].Moreover, adipose tissue produces anti-inflammatory molecules, including adiponectin, which exert beneficial effects on glucose metabolism and have the potential to reduce inflammation [12].However, the ability of anti-inflammatory molecules to counteract inflammatory response is impaired in obesity [13].Accordingly, a chronic inflammatory state has a detrimental impact on insulin resistance, dyslipidemia, hypertension, and other metabolic abnormalities associated with obesity [14].Specifically, the crosstalk between macrophages and adipocytes is crucial for initiating and sustaining a chronic inflammatory state in obese adipose tissue [15].Therefore, targeting their interaction could be an effective strategy for the treatment of obesity.
Mitochondria play a central role in energy metabolism by producing adenosine triphosphate (ATP) for the cells from food substrates, such as glucose and lipids [16].In addition to ATP generation, mitochondria are involved in biosynthesis, including the production of fatty acids [17], and in the production and elimination of reactive oxygen species (ROS) [18].In obesity, dysfunctional mitochondria can contribute to the development of metabolic abnormalities and obesity-related complications [17,18].For instance, excessive food consumption leads to elevated levels of blood glucose, resulting in the overproduction of ROS within adipocyte mitochondria [19].The increased mitochondrial ROS levels contribute to mitochondrial dysfunction, impairing their ability to perform essential metabolic processes, such as bioenergetics and biosynthesis pathways [20].Consequently, obese adipose tissues are characterized by reduced mitochondrial mass and impaired mitochondrial oxidative capacity [21].Additionally, an obesity-induced decrease in mitochondrial biogenesis further contributes to insulin resistance and low-grade inflammation [22].Therefore, strategies that promote mitochondrial biogenesis and maintain mitochondrial quality could be effective in ameliorating the progression of obesity and associated metabolic disorders.
Patulin (PAT, Figure 1A) is a mycotoxin produced by various fungal species found in diverse food products, including fruits and vegetables [23].Although toxicity studies have investigated the neurological side effects of PAT [24], some studies suggest that it possesses anticancer and anti-inflammatory effects [25,26].Notably, PAT is a potent inducer of AMPK signaling in hepatocytes, suggesting that it may have a preventive effect on the development and progression of fatty liver diseases [27].However, there is still a lack of information on the pharmacological effects of PAT on metabolic diseases, including obesity.Therefore, this study aimed to investigate the anti-inflammatory and antilipidemic properties of PAT at the cellular level.

Cell Culture
The 3T3-L1 cells were purchased from the American Type Culture Colle Manassas, VA, USA).Cells were cultured in high-glucose Dulbecco's Mod Medium (DMEM) (Welgene, Gyeongsan, Republic of Korea) containin (Welgene) and 1% penicillin-streptomycin (10,000 U/mL) solution (Gibco, C USA) at 37 °C in a 5% CO2 atmosphere.To induce differentiation, conflue incubated in DMEM containing 10% FBS (Welgene) and MDI (0.5 mM of IB DEX, and 10 µg/mL of insulin) for 2 days.The medium was replaced with taining 10% FBS and 10 µg/mL of insulin every other day.The cells were tre without PAT for 8 days during adipogenesis.
RAW 264.7 cells were obtained from the ATCC and maintained in DMEM containing 10% FBS and 1% penicillin-streptomycin (10,000 U/mL treated with 500 ng/mL of lipopolysaccharide (LPS, Sigma-Aldrich) in the absence of PAT for 24 h to induce acute inflammatory response.

Cell Viability Assay
Cell viability was assessed using MTT assay.Briefly, 3T3-L1 cells were

Cell Culture
The 3T3-L1 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).Cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) (Welgene, Gyeongsan, Republic of Korea) containing 10% BCS (Welgene) and 1% penicillin-streptomycin (10,000 U/mL) solution (Gibco, Carlsbad, CA, USA) at 37 • C in a 5% CO 2 atmosphere.To induce differentiation, confluent cells were incubated in DMEM containing 10% FBS (Welgene) and MDI (0.5 mM of IBMX, 1 µM of DEX, and 10 µg/mL of insulin) for 2 days.The medium was replaced with DMEM containing 10% FBS and 10 µg/mL of insulin every other day.The cells were treated with or without PAT for 8 days during adipogenesis.
RAW 264.7 cells were obtained from the ATCC and maintained in high-glucose DMEM containing 10% FBS and 1% penicillin-streptomycin (10,000 U/mL).Cells were treated with 500 ng/mL of lipopolysaccharide (LPS, Sigma-Aldrich) in the presence or absence of PAT for 24 h to induce acute inflammatory response.

Cell Viability Assay
Cell viability was assessed using MTT assay.Briefly, 3T3-L1 cells were seeded in 96-well plates (2 × 10 4 cells per well) containing DMEM supplemented with 10% BCS, followed by the addition of PAT to each well and culturing for 48 h.To evaluate the cytotoxicity of PAT on mature adipocytes, 3T3-L1 cells were allowed to differentiate into mature adipocytes, followed by treatment with various concentrations of PAT every 2 days.The medium was replaced with fresh medium containing insulin.MTT solution (0.5 mg/mL) was added to the incubation medium, followed by incubation for 4 h.After removing the medium, insoluble formazan crystals were dissolved in 100 µL of DMSO.The absorbance was measured at 570 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Oil Red O Staining
Lipid droplet accumulation in adipocytes was analyzed using Oil Red O (ORO, Sigma-Aldrich, St. Louis, MO, USA) staining.Briefly, cells were seeded in 24-well plates at a density of 5 × 10 4 cells/well, and differentiation was induced.Differentiated cells were washed with Dulbecco's phosphate buffered saline (DPBS, Welgene) twice and fixed with 3.7% formaldehyde solution (Biosesang, Yongin, Republic of Korea) for 15 min at 25 • C.After washing with 60% isopropanol (Ducksan, Ansan, Republic of Korea), the cells were stained with ORO solutions for 20 min at 25 • C.After removing the staining solution, the plate was washed with distilled water and dried, and cellular lipid droplets were imaged using an Olympus IX73 light microscope (Olympus, Center Valley, PA, USA).After microscopic observation, ORO staining was eluted with 100% isopropanol, and the absorbance was measured at 500 nm using a microplate reader (Molecular Devices).

Determination of TG Content
TG content was measured using a TG assay kit (#ab65336, Abcam, Cambridge, MA, USA), according to the manufacturer's protocols.Briefly, cells were washed with DPBS and resuspended in 5% NP-40/ddH2O solution.TGs were solubilized by subjecting the samples to heating and cooling three times.Thereafter, lipase was introduced to convert TG into glycerol and fatty acid.The samples were centrifuged at 15,000 rpm for 3 min to collect soluble TG, and the absorbance was measured at 570 nm using a microplate reader (Molecular Devices).Individual values were normalized to the number of cells per well.

Measurement of Glycolipid Metabolism
Glucose concentration was measured using a glucose assay kit (#ab136955, Abcam).Briefly, the amount of glucose released into the culture medium by the cells was measured by adding the reaction mixture and incubating.Glucose concentration was determined by measuring the absorbance at 450 nm.
Glycerol released from mature 3T3-L1 cells into the medium was quantified using a glycerol assay kit (#MAK117, Sigma-aldrich, St. Louis, MI, USA).Briefly, the amount of glycerol released into the medium following the various treatments was measured by adding a reaction mixture, and the absorbance was measured at 570 nm.
LDL uptake was measured using an LDL uptake assay kit (#10011125, Cayman Chemicals, Ann Arbor, MI, USA).Briefly, cells were seeded in a black plate and stained with LDL-DyLight TM solution in serum-free medium for 24 h.After removing the reaction medium, images were captured using a fluorescence microscope, and the fluorescence intensity was quantified using ImageJ (version 1.54d, NIH, Bethesda, MD, USA).

Protein Analysis
Cells were washed with DPBS and lysed with lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland).The lysate was centrifuged, and proteins were separated from lysate mixed with 5X SDS-PAGE buffer (Biosesang, Yongin, Republic of Korea) using SDS-PAGE and transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA).After treatment with a blocking buffer (Bio-Rad, Hercules, CA, USA) to prevent nonspecific binding, the membranes were incubated with primary antibodies overnight at 4 • C, followed by washing with TBST and further incubation with secondary antibodies for 1 h at 25 • C. The proteins were detected using chemiluminescent substrate (Thermo Fisher Scientific, Sunnyvale, CA, USA), and densitometry was conducted using ImageJ (version 1.54d).

RNA Analysis
Total RNA was extracted from cell pellets using an RNeasy mini kit (Qiagen, Hilden, Germany), following the manufacturer's instructions.Reverse transcription and removal of genomic DNA were conducted using a cDNA reverse transcription kit (TOY-OBO CO., LTD., Osaka, Japan).Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on a CFX ConnectTM Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA) using equal amounts of cDNA mixed with Faststart Universal SYBR Green Master (Roche) and specific primers.The PCR conditions were as follows: 95 • C for 10 min, and 40 cycles at 95 • C for 20 s, 55 • C for 30 s, and 72 • C for 30 s.The primers used for RT-qPCR are listed in Table S1.The relative mRNA levels of target genes were normalized to those of the housekeeping genes (RPLP0, β-actin, GAPDH, or Eef2), and calculated using the ∆∆Ct method.

Measurement of Mitochondrial Respiration
The real-time oxygen consumption rate (OCR) of the cells was measured using an extracellular flux analyzer (Seahorse XF, Agilent Technologies, Palo Alto, CA, USA).Briefly, cells were seeded in XF cell culture microplates and induced as described above.Thereafter, the culture medium was replaced with nonbuffered XF basal medium (#103575-100, Agilent Technologies, Santa Clara, CA, USA, XF DMEM, pH 7.4) supplemented with 25 mM of glucose, 1 mM of sodium pyruvate, and 4 mM of glutamine, after which the cells were allowed to degas in a non-CO 2 incubator at 37 • C for 1 h prior to the measurement.Cellular OCR was determined before and after sequential injection of oligomycin (1.5 µM), carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP; 1.0 µM), and antimycin A/rotenone (1.0 µM).The averaged OCR was monitored using the extracellular flux analyzer in a cycle consisting of mixing (150 s), waiting (120 s), and measuring (210 s).This cycle was repeated following each reagent injection.Mitochondrial respiration was determined by subtracting the nonmitochondrial OCR from the OCR after FCCP treatment.ATP production was determined by subtracting OCR after oligomycin treatment from the basal OCR.Finally, mitochondrial respiration data were normalized to those of the control group.

Mitochondrial Staining
Briefly, fixed cells were incubated with 200 nM of MitoTracker TM Green FM (Invitrogen) or 500 nM of MitoTracker TM Red CMXRos (Invitrogen, Waltham, MA, USA) in DPBS for 15 min at 37 • C.After washing with PBS, nuclei were stained with DAPI for 5 min, and fluorescence images were captured using a fluorescence microscope.The fluorescence intensity for mitochondrial mass and membrane potential were calculated using ImageJ (version 1.54d).

Pro-Inflammatory Cytokine and Nitric Oxide (NO) Quantification
The concentrations of pro-inflammatory cytokines (IL-6 and TNF-α) in RAW 264.7 cell culture medium were determined using BD OptEIA™ ELISA kits (BD biosciences, San Jose, CA, USA), following the manufacturer's instructions.Briefly, specific capture antibodies for each cytokine were coated on 96-well immune plates (SPL Life sciences, Pocheon, Republic of Korea) and incubated overnight at 4 • C.After rinsing off excess antibodies, assay diluent was added to inhibit nonspecific binding, followed by the addition of each cytokine standard and cell culture supernatant at the designated concentrations.After incubation and washing, captured cytokines were further incubated with a mixture of biotin-conjugated detection antibodies and streptavidin-horse radish peroxidase conjugates (sAv-HRP).Thereafter, enzymatic reaction was initiated by adding the provided substrate solution, and the reaction was stopped by applying a stop solution (1 M H 3 PO 4 ).Finally, the absorbance at 450 nm was measured using a microplate reader, and the standard curve was used to determine the cytokine concentration, expressed in ng/mL.
The concentration of NO produced in RAW 264.7 macrophages was quantified colorimetically using Promega Griess reagent (Madison, WI, USA), according to the manu-facturer's instructions.Briefly, each cell culture supernatant was mixed with 100 µL of mixed Griess reagent in a 96-well microplate and allowed to react for 10 min at 25 • C. NO concentration was determined by measuring the absorbance at 540 nm and comparing it to a standard curve established using sodium nitrite standards.

Intracellular ROS Quantification
Intracellular ROS levels were quantified using 2 ,7 -dichlorofluorescin diacetate (DCFDA) flow cytometry.After treatment with LPS in the presence or absence of PAT for 24 h, RAW 264.7 cells were further incubated with 20 µM of DCFDA solution (#ab113851, Abcam) for 30 min at 37 • C in a 5% CO 2 incubator.Thereafter, the cells were pelleted and washed with PBS two times, followed by the measurement of mean fluorescence intensity (MFI) using a CytoFLEX™ flow cytometer (Beckman Coulter, Brea, CA, USA).

Statistical Analysis
Data are presented as the mean ± standard error of the mean (SEM) of at least three replications.Statistical significance was determined using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test for multiple comparisons or unpaired twotailed Student's t-test in Prism 8 software (GraphPad Software, San Diego, CA, USA).Significant differences are indicated with different letters.Statistical significance was set at p < 0.05.

PAT Alleviates Lipid Accumulation in Mature Adipocytes
Cell viability assay was performed to elucidate the cytotoxicity of different concentrations of PAT (0.2-10 µM) on preadipocytes and mature adipocytes.Compared with that in the untreated group, PAT was not cytotoxic to preadipocytes and matured adipocytes at concentrations ≤10 µM (Figure 1B).Therefore, we investigated the effects of 1 or 5 µM of PAT on lipid accumulation during adipocyte differentiation.Adipocyte maturation is governed by insulin signaling, which promotes the uptake of glucose and free fatty acids (FFAs), leading to the accumulation of TGs [28].Thus, we investigated the effects of PAT on lipid deposition in adipocytes using ORO staining (Figure 2A).ORO staining intensity was notably higher in the differentiated group than in the undifferentiated group, indicating enhanced lipid accumulation during differentiation.Notably, PAT treatment caused a dosedependent decrease in ORO staining intensity (Figure 2B).Additionally, PAT treatment induced a dose-dependent decrease in elevated levels of TG in differentiated 3T3-L1 cells compared with that in the control group (Figure 2C).These findings suggest that PAT effectively inhibits lipid accumulation during the differentiation of 3T3-L1 preadipocytes without adversely affecting their viability.

PAT Modulates Glycolipid Metabolism in Adipocytes
To elucidate the mechanism of PAT in lipid accumulation in mature 3T3-L1 cells, we investigated changes in glucose uptake, glycerol release, and LDL uptake following PAT treatment.Specifically, the levels of glucose and glycerol in the cell culture medium were determined after treatment with PAT (1 or 5 µM).PAT treatment significantly suppressed glucose uptake in a dose-dependent manner, as evidenced by a higher glucose content in the PAT treatment group than in the untreated group (Figure 3A).Additionally, PAT treatment significantly increased glycerol concentration in the culture medium (Figure 3B) and induced a dose-dependent decrease in LDL uptake compared with that in the control group (Figure 3C).Overall, these results indicate that PAT regulates glycolipid metabolism by inhibiting glucose and LDL uptake and promoting the release of glycerol in mature adipocytes.
PAT treatment induced a dose-dependent decrease in elevated levels of ated 3T3-L1 cells compared with that in the control group (Figure 2C suggest that PAT effectively inhibits lipid accumulation during the diffe L1 preadipocytes without adversely affecting their viability.Effects of patulin (PAT) on lipid accumulation and triglyceride (TG) con adipocytes.The 3T3-L1 cells were seeded and induced to differentiate in the presen 5 µM) for 8 days.(A) Cells were stained with Oil Red O to observe lipid droplets.stained cells were extracted using isopropanol, and lipid content was quantified at 5 spectrophotometer.(C) Total TG content was measured using a TG assay kit.Data a the mean ± standard deviation (SD; n ≥ 3).Different letters indicate significantly diff p < 0.05, as determined by one-way ANOVA followed by Tukey's post hoc test.

PAT Modulates Glycolipid Metabolism in Adipocytes
To elucidate the mechanism of PAT in lipid accumulation in mature 3T investigated changes in glucose uptake, glycerol release, and LDL uptake fo treatment.Specifically, the levels of glucose and glycerol in the cell culture m determined after treatment with PAT (1 or 5 µM).PAT treatment significantl glucose uptake in a dose-dependent manner, as evidenced by a higher gluco the PAT treatment group than in the untreated group (Figure 3A).Additional ment significantly increased glycerol concentration in the culture medium (F induced a dose-dependent decrease in LDL uptake compared with that i group (Figure 3C).Overall, these results indicate that PAT regulates glycol lism by inhibiting glucose and LDL uptake and promoting the release of gl ture adipocytes.

PAT Suppresses the Expression of Adipogenic/Lipogenic Genes
Furthermore, we investigated the effect of PAT on the expression of ad lipogenic mediators in 3T3-L1 adipocytes.There was a significant increas FASN, and ACLY protein expression levels following the differentiation of 3 ipocytes into mature adipocytes (Figure 4A).However, PAT treatment sig

PAT Suppresses the Expression of Adipogenic/Lipogenic Genes
Furthermore, we investigated the effect of PAT on the expression of adipogenic and lipogenic mediators in 3T3-L1 adipocytes.There was a significant increase in PPARγ, FASN, and ACLY protein expression levels following the differentiation of 3T3-L1 preadipocytes into mature adipocytes (Figure 4A).However, PAT treatment significantly decreased the expression of the proteins in a dose-dependent manner (Figure 4B).Additionally, RT-qPCR showed that PAT treatment significantly suppressed the upregulation of key adipogenic/lipogenic genes, including Pparγ, C/ebpα, Fabp4, Cd36, Dgat2, and Gpat, during the differentiation process compared with that in the control group (Figure 4C).These results indicate that PAT exerts an inhibitory effect on the expression of adipogenic and lipogenic genes.

PAT Increases the Expression of Fatty Acid Oxidation-Related Genes
To evaluate the effect of PAT on fatty acid oxidation (FAO), we examined sion of PGC1α and related molecules at the protein and mRNA levels.Com that in undifferentiated 3T3-L1 adipocytes, there was a decrease in PGC1α protein expression in matured adipocytes.However, treatment with 5 µM of lated PGC1α expression (Figure 5A-C) and significantly increased the express gene compared with that in untreated control group (Figure 5D).Overall, t

PAT Increases the Expression of Fatty Acid Oxidation-Related Genes
To evaluate the effect of PAT on fatty acid oxidation (FAO), we examined the expression of PGC1α and related molecules at the protein and mRNA levels.Compared with that in undifferentiated 3T3-L1 adipocytes, there was a decrease in PGC1α mRNA and protein expression in matured adipocytes.However, treatment with 5 µM of PAT stimulated PGC1α expression (Figure 5A-C) and significantly increased the expression of Cpt1a gene compared with that in untreated control group (Figure 5D).Overall, these results suggest that PAT inhibits adipogenesis by promoting FAO.

PAT Enhances Mitochondrial Function in Adipocytes
PGC1α plays a pivotal role in mitochondrial metabolism, promoting biogenesis and bioenergetics [29].Therefore, we investigated the effects o on mitochondrial respiration in mature 3T3-L1 adipocytes by measuring horse analysis.Notably, treatment with 5 µM of PAT significantly increas compared with that in the untreated group (Figure 6A,B).To further eluci of PAT on mitochondrial integrity, we examined changes in mitochon membrane potential using MitoTracker TM Green and Red staining, resp 6C-F).PAT-treated cells exhibited more intense green staining compared cells, indicative of an increase in mitochondrial mass in mature adipocyte Additionally, PAT treatment increased mitochondrial membrane potentia pocytes.Moreover, flow cytometry confirmed that PAT treatment not only chondrial mass but also improved mitochondrial membrane integrity (Fig tively , these results indicate that PAT positively influences mitochondrial f proving both mitochondria mass and quality in mature adipocytes.

PAT Enhances Mitochondrial Function in Adipocytes
PGC1α plays a pivotal role in mitochondrial metabolism, promoting mitochondrial biogenesis and bioenergetics [29].Therefore, we investigated the effects of PAT treatment on mitochondrial respiration in mature 3T3-L1 adipocytes by measuring OCR using Seahorse analysis.Notably, treatment with 5 µM of PAT significantly increased cellular OCR compared with that in the untreated group (Figure 6A,B).To further elucidate the impact of PAT on mitochondrial integrity, we examined changes in mitochondrial mass and membrane potential using MitoTracker TM Green and Red staining, respectively (Figure 6C-F).PAT-treated cells exhibited more intense green staining compared with untreated cells, indicative of an increase in mitochondrial mass in mature adipocytes (Figure 6C,D).Additionally, PAT treatment increased mitochondrial membrane potential in mature adipocytes.Moreover, flow cytometry confirmed that PAT treatment not only elevated mitochondrial mass but also improved mitochondrial membrane integrity (Figure S1).Collectively, these results indicate that PAT positively influences mitochondrial functions by improving both mitochondria mass and quality in mature adipocytes.

PAT Inhibits the Secretion of Pro-Inflammatory Cytokines in Macrophages
Macrophages are crucial immune cells involved in obesity-associated chronic lowgrade inflammation [30].In addition to its antilipidemic effect in 3T3-L1 adipocytes, we investigated the effect of PAT in inflammatory response in LPS-treated RAW 264.7 macrophages.MTT assay demonstrated that PAT was not cytotoxic to RAW 264.7 cells at concentrations ≤2 µM (Figure S2).Additionally, PAT treatment significantly suppressed LPS-induced transcription of the pro-inflammatory Tnf-α and Il-6 genes in a dose-dependent manner (Figure 7A,B).Moreover, the LPS-induced increase in the secretion of both IL-6 and TNF-α was significantly inhibited by PAT treatment in a dose-dependent manner compared with that in the untreated group (Figure 7C,D).Overall, these results indicate that PAT reduces pro-inflammatory cytokine production in LPS-induced macrophages.

PAT Reduces Inflammatory Mediator Production in Macrophages
Furthermore, we investigated the effects of PAT on inflammatory mediators, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).PAT treatment at concentrations ≥ 1 µM significantly inhibited LPS-induced upregulation in the mRNA expression of inos and Cox-2 (Figure 8A,B).Moreover, PAT treatment significantly suppressed the LPS-induced increase in the production of nitric oxide (NO), a byproduct of inos, in a dose-dependent manner compared with that in the untreated group (Figure 8C).These findings suggest that PAT may improve inflammatory response by inhibiting the production of pro-inflammatory cytokines and mediators in LPS-stimulated macrophages.

PAT Reduces Inflammatory Mediator Production in Macrophages
Furthermore, we investigated the effects of PAT on inflammatory mediators, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).PAT treatment at concentrations ≥1 µM significantly inhibited LPS-induced upregulation in the mRNA expression of inos and Cox-2 (Figure 8A,B).Moreover, PAT treatment significantly suppressed the LPS-induced increase in the production of nitric oxide (NO), a byproduct of inos, in a dose-dependent manner compared with that in the untreated group (Figure 8C).These findings suggest that PAT may improve inflammatory response by inhibiting the production of pro-inflammatory cytokines and mediators in LPS-stimulated macrophages.

PAT Reduces Inflammatory Mediator Production in Macrophages
Furthermore, we investigated the effects of PAT on inflammatory mediators, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).PAT treatment at concentrations ≥ 1 µM significantly inhibited LPS-induced upregulation in the mRNA expression of inos and Cox-2 (Figure 8A,B).Moreover, PAT treatment significantly suppressed the LPS-induced increase in the production of nitric oxide (NO), a byproduct of inos, in a dose-dependent manner compared with that in the untreated group (Figure 8C).These findings suggest that PAT may improve inflammatory response by inhibiting the production of pro-inflammatory cytokines and mediators in LPS-stimulated macrophages.

PAT Modulates Mitochondrial Function in Macrophage
The mitochondrial energy metabolic phenotype of macrophages plays a crucial role in shaping their function and immunophenotype [31].Thus, we investigated the effects of PAT on mitochondrial respiration in macrophages by measuring the OCR.LPS stimulation decreased mitochondrial respiration and ATP production in macrophages considerably (Figure S3).Notably, PAT treatment at concentrations ≥1.0 µM enhanced both mitochondrial respiration (Figure 9A,B) and ATP production (Figure S4).Furthermore, flow cytometry analysis was performed to assess intracellular ROS production, mitochondrial mass, and mitochondrial membrane potential (Figure 9C-F).PAT treatment significantly reversed LPS-induced increase in ROS production in a dose-dependent manner (Figure 9D) and significantly increased mitochondrial mass compared with that in the untreated LPSstimulated group (Figure 9E).However, PAT treatment did not significantly affect mitochondrial membrane potential in LPS-stimulated macrophages (Figure 9F).Collectively, these results suggest that the enhancement of mitochondrial bioenergetics contributes to the anti-inflammatory effect of PAT, likely mediated by its antioxidant properties.

PAT Modulates Mitochondrial Function in Macrophage
The mitochondrial energy metabolic phenotype of macrophages plays a crucial role in shaping their function and immunophenotype [31].Thus, we investigated the effects of PAT on mitochondrial respiration in macrophages by measuring the OCR.LPS stimulation decreased mitochondrial respiration and ATP production in macrophages considerably (Figure S3).Notably, PAT treatment at concentrations ≥ 1.0 µM enhanced both mitochondrial respiration (Figure 9A,B) and ATP production (Figure S4).Furthermore, flow cytometry analysis was performed to assess intracellular ROS production, mitochondrial mass, and mitochondrial membrane potential (Figure 9C-F).PAT treatment significantly reversed LPS-induced increase in ROS production in a dose-dependent manner (Figure 9D) and significantly increased mitochondrial mass compared with that in the untreated LPS-stimulated group (Figure 9E).However, PAT treatment did not significantly affect mitochondrial membrane potential in LPS-stimulated macrophages (Figure 9F).Collectively, these results suggest that the enhancement of mitochondrial bioenergetics contributes to the anti-inflammatory effect of PAT, likely mediated by its antioxidant properties.

Discussion
Research evidence suggests that chronic obesity is accompanied by the accumulation of macrophages, contributing to the development of insulin resistance in adipose tissue, skeletal muscle, and the liver [32].Despite this understanding, the exact cause of inflammation in obesity remains poorly understood, and effective targeted therapeutic agents are limited.Currently, lifestyle improvements, such as changes in diet and exercise, are commonly adopted strategies to combat obesity.However, achieving and maintaining these behavioral changes can be challenging, making drug options a consideration [33].Although various antiobesity drugs are currently available, their long-term use can lead to serious side effects [34].Thus, studies are necessary to establish a link between the consumption of safe natural food ingredients and a decrease in obesity and related complications [35].Research evidence indicates that the consumption of natural products is associated with reduced risk of obesity-related complications [36].Given the increasing recognition that safe alternatives derived from natural sources may be effective in alleviating obesity, extensive research is necessary to identify natural compounds with antiobesity effects.Therefore, this study elucidated the antilipidemic and anti-inflammatory effects of PAT, a natural compound derived from fungi, through in vitro experiments.
Adipose tissues are insulin-sensitive peripheral tissues, secreting various hormones to maintain overall energy homeostasis in the body [37].The regulation of adipogenesis entails an intricate transcriptional cascade that includes the sequential activation of C/EBP and PPARγ at the molecular level [38].In adipocytes, PPARγ and C/EBPα act synergistically to promote the synthesis and transport of FFAs by sharing binding sites [38].This synergistic action is essential for lipid metabolism and proper functioning of adipose tissue in maintaining energy balance [39].Additionally, both PPARγ and C/EBPα are essential for driving insulin-stimulated glucose uptake and can induce excessive production of FFAs in obese individuals [40].Therefore, regulating transcriptional networks in differentiating adipocytes could be a promising therapeutic strategy for FFA-induced insulin resistance and obesity.In the present study, PAT treatment decreased the protein and mRNA expression of PPARγ and C/EBPα in differentiated 3T3-L1 cells, a commonly used model for studying adipocyte differentiation.Additionally, PAT treatment suppressed insulin-induced upregulated of lipogenic genes, glucose and LDL uptake, lipid droplet accumulation, and TG levels, suggesting that PAT can influence adipogenesis and lipogenesis by modulating the expression of key transcription factors involved in insulin-induced adipocyte differentiation.However, further studies are necessary to comprehensively understand the specific molecular pathways through which PAT inhibits lipid accumulation in mature adipocytes.
During periods of increased energy demand, TGs stored in adipose tissues are broken down into FFAs and glycerol, which serve as energy for other organs [41].This process involves the proportional release of glycerol and FFAs from adipocytes, making glycerol release from mature adipocytes an important index for assessing lipolysis [41].FFAs are further oxidized to provide energy through mitochondrial FAO, and the PGC1-α plays a crucial role in activating mitochondrial biogenesis and FAO [42].Additionally, the transcriptional activity of PGC-1α is responsible for regulating the expression of gene networks that control glucose consumption, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) in adipose tissue [43].Considering that mitochondrial dysfunction in adipocytes is closely associated with insulin resistance in obese individuals [44], enhancing mitochondrial function through the activation of PGC1-α could be a promising strategy to prevent obesity and related metabolic complications.
Several studies have demonstrated that natural compounds can exert protective effects against obesity by activating PGC1-α.For instance, zeaxanthin, a carotenoid found in various vegetables, enhanced PGC1-α expression and mitochondrial respiration, leading to decreased lipid accumulation in 3T3-L1 adipocytes [45].Additionally, ginsenoside Rb1 increased PGC1-α expression and mitochondrial oxidative function, including ATP generation, in mature adipocytes, thereby improving glycolipid metabolism and reducing weight gain in obese mice [46].Moreover, sesamol, the main phenolic compound derivative in sesame oil, suppressed high-fructose-diet-induced lipogenesis and bodyweight gain by upregulating mitochondrial metabolism-related genes, including PGC1-α, in mature adipocytes [47].In the present study, treatment with 5 µM of PAT significantly elevated glycerol secretion and increased the protein and mRNA expression of PGC1-α and its FAO downstream target gene Cpt1a in differentiated 3T3-L1 adipocytes, indicating an induction of the breakdown of FFAs.Moreover, PAT treatment had a notable impact on mitochondrial function by significantly enhancing mitochondrial oxidative capacity, increasing mitochondrial mass, and improving mitochondrial membrane integrity.Overall, these findings suggest that PAT has the potential to improve mitochondrial function.Based on its positive effects on mitochondrial integrity, PAT could serve as a promising therapeutic for ameliorating obesity and its associated metabolic dysregulations.
Adipose tissue, a substantial endocrine organ harboring several immune cells, particularly macrophages, plays a crucial role in both health and disease conditions by clearing apoptotic adipocytes and facilitating tissue remodeling to maintain homeostasis [48].In the obese state, the population of macrophages in adipose tissue increases by more than three times compared with that in the lean state, and these macrophages interact with adipocytes, leading to an inflammatory phenotype in obese individuals [49].During this process, adipocytes detect danger signals mimicking microbial infections and trigger inflammatory responses by activating the toll-like receptor (TLR) signaling pathway, resulting in the release of pro-inflammatory mediators, such as NO and cytokines [50].Notably, the TNF-α level was upregulated in the adipose tissue of obese subjects, and the inhibition of TNF-α expression was closely associated with weight loss in these individuals [9].Therefore, downregulating inflammatory cytokines and mediators produced by activated macrophages could be a beneficial approach for preventing obesity-related chronic inflammation.In the present study, RAW 264.7 cells were stimulated with LPS to induce oxidative stress and inflammatory signaling cascade through TLRs on macrophages [51], and the antiinflammatory effect of PAT was evaluated.Consistent with our data, PAT demonstrated the potential to suppress NO production by downregulating mitogen-activated protein kinase (MAPK) signaling pathway in LPS-induced murine peritoneal macrophages [26].Additionally, PAT treatment inhibited IL-6 and TNF-α expression, as well as the expression of inducible enzyme-encoding genes, such as Cox-2, under conditions of acute inflammation.
Recent research findings suggest a close relationship between mitochondrial functions and the development and progression of metabolic diseases, including obesity [52].Moreover, the intricate interplay of metabolic and immune responses, regulated by macrophage mitochondria, has been emphasized due to the crucial role of these responses in assessing the inflammatory states [31].In physiological conditions, healthy mitochondria maintain a balance between the formation and elimination of ROS through their antioxidant defense systems [53].However, prolonged inflammatory conditions can alter the equilibrium between antioxidants and free radicals, leading to abnormal ROS generation, which triggers an increase in the production of pro-inflammatory mediators and contributes to oxidative stress and development of the chronic state [54].Moreover, overnutrition is associated with chronic oxidative and inflammatory stress in individuals with obesity [55], suggesting that inhibiting excessive ROS production by maintaining mitochondrial oxidative function could be a promising therapeutic approach to alleviate obesity-related inflammatory diseases.In this study, PAT treatment increased mitochondrial respiration, ATP production, and mitochondrial mass, and it suppressed intracellular ROS generation in LPS-stimulated macrophages.Typically, mitochondrial mass and ROS generation exhibit a proportional relationship.However, the discrepancy in our results might be attributed to PAT-induced improvement in antioxidant capacity via upregulation of mitochondrial biogenesis.This notion is supported by the upregulation of the PGC1α gene expression in PAT-treated 3T3-L1 adipocytes.Consistent with our findings, a previous study demonstrated that PGC1α enhanced both mitochondrial mass and the expression of primary mitochondrial antioxidant enzymes, such as SOD2 [56].Overall, these findings suggest that PAT exerts

Figure 1 .
Figure 1.Effects of patulin (PAT) on the viability of 3T3-L1 adipocytes.(A) Chemic PAT.(B) The 3T3-L1 preadipocytes were treated with different concentrations of PAT bar graph).The 3T3-L1 preadipocytes were differentiated in a medium containing M ence of different concentrations of PAT for 8 days (right bar graph).Cell viability using a WST-1 kit.

Figure 1 .
Figure 1.Effects of patulin (PAT) on the viability of 3T3-L1 adipocytes.(A) Chemical structure of PAT.(B) The 3T3-L1 preadipocytes were treated with different concentrations of PAT (0-10 µM, left bar graph).The 3T3-L1 preadipocytes were differentiated in a medium containing MDI in the presence of different concentrations of PAT for 8 days (right bar graph).Cell viability was measured using a WST-1 kit.

Figure 2 .
Figure 2. Effects of patulin (PAT) on lipid accumulation and triglyceride (TG) content in 3T3-L1 adipocytes.The 3T3-L1 cells were seeded and induced to differentiate in the presence of PAT (1 or 5 µM) for 8 days.(A) Cells were stained with Oil Red O to observe lipid droplets.(B) Oil Red O-stained cells were extracted using isopropanol, and lipid content was quantified at 550 nm using a spectrophotometer.(C) Total TG content was measured using a TG assay kit.Data are presented as the mean ± standard deviation (SD; n ≥ 3).Different letters indicate significantly different values at p < 0.05, as determined by one-way ANOVA followed by Tukey's post hoc test.

Figure 3 .
Figure 3.Effect of patulin (PAT) on glycolipid metabolism.The 3T3-L1 cells were cu duced to differentiate in the presence of PAT (1 or 5 µM) for 8 days.(A,B) The cultur collected, and the amount of glucose and glycerol in the medium was measured.(C was examined under a fluorescence microscope.The relative fluorescence intensit quantified using ImageJ (version 1.54d).Data are presented as the mean ± standard n = 3).Different letters indicate significantly different values at p < 0.05, as determin way ANOVA followed by Tukey's post hoc test.

Figure 3 .
Figure 3.Effect of patulin (PAT) on glycolipid metabolism.The 3T3-L1 cells were cultured and induced to differentiate in the presence of PAT (1 or 5 µM) for 8 days.(A,B) The culture medium was collected, and the amount of glucose and glycerol in the medium was measured.(C) LDL uptake was examined under a fluorescence microscope.The relative fluorescence intensity per cell was quantified using ImageJ (version 1.54d).Data are presented as the mean ± standard deviation (SD; n = 3).Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.

Figure 4 .
Figure 4. Effects of patulin (PAT) on the expression of lipid-metabolism-related prot metabolic regulators in 3T3-L1 adipocytes.The 3T3-L1 cells were seeded and induced ate in the presence of PAT (1 or 5 µM) for 8 days.(A) Protein expression levels of PPAR ACLY were analyzed using immunoblotting assay.(B) Bar graphs indicate the densito tification of PPARγ/α-tubulin, FASN/α-tubulin, and ACLY/α-tubulin bands.Data are the mean ± standard deviation (SD; n = 3).(C) mRNA expression of genes related to adi pogenesis in 3T3-L1 adipocytes were analyzed using RT-qPCR.Data are expressed a SD (n = 3).Different letters within each column indicate significant differences at p between treatments, as determined by one-way ANOVA followed by Tukey's post hoc

Figure 4 .
Figure 4. Effects of patulin (PAT) on the expression of lipid-metabolism-related proteins and key metabolic regulators in 3T3-L1 adipocytes.The 3T3-L1 cells were seeded and induced to differentiate in the presence of PAT (1 or 5 µM) for 8 days.(A) Protein expression levels of PPARγ, FASN, and ACLY were analyzed using immunoblotting assay.(B) Bar graphs indicate the densitometric quantification of PPARγ/α-tubulin, FASN/α-tubulin, and ACLY/α-tubulin bands.Data are expressed as the mean ± standard deviation (SD; n = 3).(C) mRNA expression of genes related to adipogenesis/lipogenesis in 3T3-L1 adipocytes were analyzed using RT-qPCR.Data are expressed as the mean ± SD (n = 3).Different letters within each column indicate significant differences at p < 0.05 levels between treatments, as determined by one-way ANOVA followed by Tukey's post hoc test (B,C).

Figure 5 .
Figure 5. Effects of patulin (PAT) on the expression of fatty acid oxidation-related adipocytes.The 3T3-L1 cells were cultured and induced to differentiate in the pre 5 µM) for 8 days.(A) Protein expression levels of PGC1α were determined by imm The bar graph indicates the relative intensity of PGC1α/ β-Actin bands.mRNA ex (C) Pgc1α and (D) Cpt1a were analyzed using RT-qPCR.Data are expressed as the deviation (SD; n = 3).Different letters indicate significantly different values at p < 0. by one-way ANOVA followed by Tukey's post hoc test.

Figure 5 .
Figure 5. Effects of patulin (PAT) on the expression of fatty acid oxidation-related genes in 3T3-L1 adipocytes.The 3T3-L1 cells were cultured and induced to differentiate in the presence of PAT or 5 µM) for 8 days.(A) Protein expression levels of PGC1α were determined by immunoblotting.(B) The bar graph indicates the relative intensity of PGC1α/ β-Actin bands.mRNA expression levels of (C) Pgc1α and (D) Cpt1a were analyzed using RT-qPCR.Data are expressed as the mean ± standard deviation (SD; n = 3).Different letters indicate significantly different values at p < 0.05, as determined by one-way ANOVA followed by Tukey's post hoc test.

Figure 6 .
Figure 6.Effects of patulin (PAT) on mitochondrial oxidative function in 3T3-L1 adipocy 3T3-L1 cells were seeded and induced to differentiate in the presence of PAT (1 or 5 µM) fo (A,B) Oxygen consumption rate (OCR) was measured as described in the Materials and section.Representative data from three independent experiments are presented.The indic ues were normalized to the protein content determined using BCA assay.(B) Different lett cate significantly different values at p < 0.05, as determined using one-way ANOVA foll Tukey's post hoc test.(C,E) Representative images of MitoTracker Green-stained adipocyte mitochondrial mass) and CMXRos-stained adipocytes (E, for mitochondrial membrane p ΔΨm) are shown.(D,F) Quantitative analysis of fluorescence intensity is shown.Statistica cance (p value) was determined using unpaired two-tailed Student's t-test.

Figure 6 .
Figure 6.Effects of patulin (PAT) on mitochondrial oxidative function in 3T3-L1 adipocytes.The 3T3-L1 cells were seeded and induced to differentiate in the presence of PAT (1 or 5 µM) for 8 days.(A,B) Oxygen consumption rate (OCR) was measured as described in the Materials and Methods section.Representative data from three independent experiments are presented.The indicated values were normalized to the protein content determined using BCA assay.(B) Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.(C,E) Representative images of MitoTracker Green-stained adipocytes (C, for mitochondrial mass) and CMXRos-stained adipocytes (E, for mitochondrial membrane potential, ∆Ψm) are shown.(D,F) Quantitative analysis of fluorescence intensity is shown.Statistical significance (p value) was determined using unpaired two-tailed Student's t-test.

Figure 7 .
Figure 7. Effects of patulin (PAT) on pro-inflammatory cytokine production in lipopolysaccharide (LPS)-induced macrophages.RAW 264.7 cells were preincubated and further exposed to LPS (500 ng/mL) in the presence of PAT (1 or 5 µM) for 24 h.(A,B) The transcription of pro-inflammatory genes (Il-6 and Tnf-α) in RAW 264.7 cells was assessed using RT-qPCR.(C,D) The production of proinflammatory cytokines (IL-6 and TNF-α) in cell supernatants was quantified using ELISA.Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.

Figure 8 .
Figure 8. Effects of patulin (PAT) on inflammatory gene transcription and inflammatory mediator production in lipopolysaccharide (LPS)-induced macrophages.(A,B) mRNA expression of inos and Cox-2 in RAW 264.7 cells were analyzed using RT-qPCR.(C) The production of nitric oxide (NO) was quantified by colorimetric assay using Griess reagent.Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.

Figure 7 .
Figure 7. Effects of patulin (PAT) on pro-inflammatory cytokine production in lipopolysaccharide (LPS)-induced macrophages.RAW 264.7 cells were preincubated and further exposed to LPS (500 ng/mL) in the presence of PAT (1 or 5 µM) for h.(A,B) The transcription of pro-inflammatory genes (Il-6 and Tnf-α) in RAW 264.7 cells was assessed using RT-qPCR.(C,D) The production of pro-inflammatory cytokines (IL-6 and TNF-α) in cell supernatants was quantified using ELISA.Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.

Antioxidants 2023 , 17 Figure 7 .
Figure 7. Effects of patulin (PAT) on pro-inflammatory cytokine production in lipopolysaccharide (LPS)-induced macrophages.RAW 264.7 cells were preincubated and further exposed to LPS (500 ng/mL) in the presence of PAT (1 or 5 µM) for 24 h.(A,B) The transcription of pro-inflammatory genes (Il-6 and Tnf-α) in RAW 264.7 cells was assessed using RT-qPCR.(C,D) The production of proinflammatory cytokines (IL-6 and TNF-α) in cell supernatants was quantified using ELISA.Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.

Figure 8 .
Figure 8. Effects of patulin (PAT) on inflammatory gene transcription and inflammatory mediator production in lipopolysaccharide (LPS)-induced macrophages.(A,B) mRNA expression of inos and Cox-2 in RAW 264.7 cells were analyzed using RT-qPCR.(C) The production of nitric oxide (NO) was quantified by colorimetric assay using Griess reagent.Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.

Figure 8 .
Figure 8. Effects of patulin (PAT) on inflammatory gene transcription and inflammatory mediator production in lipopolysaccharide (LPS)-induced macrophages.(A,B) mRNA expression of inos and Cox-2 in RAW 264.7 cells were analyzed using RT-qPCR.(C) The production of nitric oxide (NO) was quantified by colorimetric assay using Griess reagent.Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.

Figure 9 .
Figure 9. Effects of patulin (PAT) on mitochondrial oxidative function in lipopolysaccharide (LPS)induced RAW 264.7 macrophages.(A,B) Oxygen consumption rate (OCR) was measured as described in the Materials and Methods section.Representative data from three independent experiments are presented.The indicated values were normalized to the protein content, determined using BCA assay.(C) Flow cytometry was performed to examine macrophages stained with DCFDA, Mi-toTracker Green, and MitoTracker Red CMXRos.(D) Intracellular ROS levels, (E) mitochondria (Mito) mass, and (F) Mito membrane potential were determined using mean fluorescence intensity (MFI) obtained through flow cytometry, employing specific probes for staining.The data were normalized to the values of LPS-treated control.Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.

Figure 9 .
Figure 9. Effects of patulin (PAT) on mitochondrial oxidative function in lipopolysaccharide (LPS)induced RAW 264.7 macrophages.(A,B) Oxygen consumption rate (OCR) was measured as described in the Materials and Methods section.Representative data from three independent experiments are presented.The indicated values were normalized to the protein content, determined using BCA assay.(C) Flow cytometry was performed to examine macrophages stained with DCFDA, MitoTracker Green, and MitoTracker Red CMXRos.(D) Intracellular ROS levels, (E) mitochondria (Mito) mass, and (F) Mito membrane potential were determined using mean fluorescence intensity (MFI) obtained through flow cytometry, employing specific probes for staining.The data were normalized to the values of LPS-treated control.Different letters indicate significantly different values at p < 0.05, as determined using one-way ANOVA followed by Tukey's post hoc test.